The long-term goals of this proposal is to understand how the various Cdc2 regulatory mechanisms contribute to the control of cell cycle progression in vertebrates. Entry into mitosis is controlled by the Cdc2 cyclin dependent kinase. Proper regulation of Cdc2 ensures that mitosis occurs only after earlier phases of the cell cycle have been completed. This strict control is largely post-translational with inhibitory phosphorylation playing a key role. In metazoans, two types of inhibitory Cdc2 kinases have been identified: the soluble Wee1 found in the nucleus and the membrane-associated Myt1 found in the cytoplasm. Understanding the regulation of Wee1 and Myt1 is required to understand how cell proliferation is controlled. While recent studies have begun to unravel the regulatory mechanisms modulating Wee1, very little is known about Myt1. Myt1 may play a unique role in cell cycle control. For example, because of its localization to the cytoplasm, Myt1 is the only inhibitory kinase available to directly inhibit the interphase pool of the Cdc2/Cyclin B complex. Furthermore, Myt1 is the only Cdc2 inhibitory kinase found in oocytes. Oocytes must remain arrested in G2 with inactive Cdc2 for proper development. Understanding the regulation of Myt1 will aid in our understanding of these processes and give us insights into the general mechanisms used to control cell division and to prevent disease such as cancer. The goals of this proposal are to understand how Myt1 is regulated to ensure proper progression through the cell cycle. To accomplish these goals, we will: (1) Characterize the regulatory pathways controlling Myt1. We will examine the regulation of Myt1 during the cell cycle, oocyte maturation, and checkpoint response. (2) Map the cis-acting regulatory domains of Myt1. To aid in our understanding of Myt1 regulation and cell cycle control, we will determine the regions of Myt1 required for activity as well as the regions responsible for regulation. (3) Identify and characterize trans-acting regulators of Myt1. In order to understand at the molecular level how the initiation of mitosis is signaled and triggered, we need to understand the upstream regulators of Myt1. We will use both genetic and biochemical approaches to identify and characterize the regulators of Myt1.